Based upon results of both our clinical trial of IGFIR antibody treatment in Ewing's sarcoma and rhabdomyosarcoma patients showing rapid development of acquired resistance among responsive patients with most responding patients developing progressive disease within 5 months of treatment, we have focused on studying mechanisms of resistance in the laboratory. We have already published data showing that rhabdomyosarcoma mouse xenografts develop similar resistance to IGFIR antibody treatment within 60 days. We have now demonstrated that at least 2 mechanisms of resistance are observed. First we found the Src family kinase (SFK) member YES is rapidly activated in rhabdomyosarcoma cell lines and xenografts after exposure to IGFIR antibody. Second we showed that YES is highly expressed in a subset of rhabdomyosarcoma human tumors. Third we showed that simultaneous blockade of SFK with dasatinib or saracatinib leads to enhanced tumor inhibition when combined with IGFIR blockade. These data are currently under revison for publication. We would like to pursue a potential clinical trial testing the combination of SFK and IGFIR inhibition in recurrent rhabdomyosarcoma. We have also established cell lines resistant to IGFIR antibody treatment and used these cell lines to look for phospho-proteomic differences between the resistant and sensitive parental cell lines. We identified PDGFR-beta as being markedly up-regulated in the resistant cell line and have confirmed that concomitant inhibition of PDGFR and IGFIR leads to enhanced cell killing of these cell lines in vitro. Ongoing experiments seek to determine whether this synergy will be seen using human xenograft studies in immundeficient mice. We have completed a kinome siRNA screen in Ewing's sarcoma cells in collaboration with Natasha Caplen. We have identified 11 candidate kinases that appear to be necessary for Ewing's sarcoma cell growth including Aurora kinase B, Chek1, and Polo-like kinase. Of further interest, the importance of several of these kinases were confirmed in an independent high-throughput compound screen against 63 human sarcoma cell lines including 19 Ewing's sarcoma cell lines, in collaboration with Dr. Bev Teicher's group. This screen independently demonstrated that Ewing's sarcoma cells were particularly sensitive to inhibitors of Aurora kinase, Chek1/2, and Polo-like kinase. We are currently focusing on understanding the mechanism of sensitivity to Aurora kinase inhibitors and are testing these inhibitors in xenograft studies. We have further characterized the largest series of SDH deficient GIST tumors and have shown that about 50% are germline mutations, while another group have specific alterations in the promoter region of the SDHC gene leading to loss of expression. We confirmed that all the SDH deficient tumors are characterized global hypermethylation, suggesting that the SDH mutations are correlated with an inability to demethylate DNA. These finding have recently been published in Cancer Discovery.